The biochemical mechanisms involved in the action of antidiuretic homone (ADH) are not known. It is widely assumed that cAMP-dependent protein phosphorylation is intimately involved in the physiological response to ADH. We plan to evaluate this hypothesis at three known or presumed sites for ADH action in its target cells. 1) ADH-receptor interactions: We have described the characteristics of (3H) (8-lysine) vasopressin binding for the first time in intact cells utilizing a porcine kidney cell culture model, LLC-PK1. Hormonal binding is modulated by hypertonic NaCl and extracellular ATP. We will determine if this regulation reflects the phosphorylation of the ADH receptor or some component of the adenylyl cyclase complex by an exokinase or intracellular protein kinase. 2) Type II cAMP-dependent protein kinase (cAMP-PK): A 50,000 mol wt phosphoprotein previously identified in toad bladder may be the phosphoregulatory unit (RII) of the type II cAMP-PK. We will evaluate this possibility utilizing a photoaffinity label of cAMP and affinity chromatography. Since the state of phosphorylation of RII determines in part the cAMP-PK activity, we will evaluate the role of hypertonic NaCl and protein phosphatase in regulating phospho-RII in toad bladder and LLC-PKl cells. 3) The integrity of microtubules and microfilaments is essential for ADH to elicit a physiological response. We will determine if phosphorylated microtubule-associated proteins (MAP) and actin-binding proteins (ABP) exist in toad bladder, and whether their phosphorylation is altered by ADH. Phospho-MAP and phospho-ABP will be evaluated in vitro by their ability to stimulate tubulin polymerization and actin gelation, respectively. The interaction of actin and myosin is regulated by myosin light chain (MLC) kinase. We will determine if ADH inhibits MLC kinase by phosphorylation in toad bladder, resulting in relaxation of the apical membrane. Conversely, ADH stimulates the contraction of cloned glomerular mesangial cells which should result in dephosphorylation of MLC kinase. These studies should provide the first evidence for ADH-stimulated phosphorylation of sub-strates with known physiological activity.